Common fragile sites (CFSs) are large chromosomal regions that exhibit gaps and breaks on metaphase chromosomes upon replication stress. They are unstable at early stages of cancer development and are often associated with chromosomal rearrangements in cancer. Since CFSs are part of the normal chromosomes, it is important to understand how CFSs are protected to maintain genome stability, thereby preventing cancer development. In this study, we propose to investigate the underlying mechanisms of how DNA damage checkpoint and DNA double-strand break (DSB) repair networks function to preserve genome integrity at CFSs. First, we will study the role of the replication machinery and replication checkpoints in the protection of stalled replication forks at CFSs. We will also investigate the involvement of replication fork regression in removing DNA secondary structures at CFSs and protecting for fork stability. Second, we will use our newly established EGFP-based repair substrates to study the pathways that are utilized to repair DSBs associated with CFSs. Since pathway choice would influence repair fidelity and genome stability at CFSs, understanding the DSB repair mechanisms to protect CFSs is extremely important for revealing the cause of cancer-related DNA lesions at CFSs. Third, oncogene expression induces CFS instability, but it remains unknown how such instability leads to cancer-prone DNA lesions. We will study the types of DNA lesions formed upon oncogene expression, and probe the roles of DNA repair and the checkpoint network in the prevention of oncogene-induced genome instability at CFSs. These studies will significantly help understand the mechanisms of cancer development that is associated with CFSs, and provide insight into cancer etiology. They will also help develop novel therapeutic strategies to prevent CFS-associated tumorigenesis.